Canola event PV-BNGT04(RT73) and compositions and methods for detection thereof

ABSTRACT

The present invention provides assays for detecting the presence of the PV-BNGT04(RT73) canola event based on the DNA sequence of the recombinant construct inserted into the canola genome and of genomic sequences flanking the insertion site.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/415,305,filed Apr. 28, 2003, issued as U.S. Pat. No. 7,306,909, which is a §371application of PCT/US2001/048583, filed Oct. 22, 2001, which claimsbenefit of priority from U.S. Provisional Patent Application No.60/244,346, filed Oct. 30, 2000.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more specifically the invention relates to transgenic glyphosatetolerance in a plant. The invention more specifically relates to aglyphosate tolerant canola plant PV-BNGT04(RT73) and to assays fordetecting the presence of canola plant PV-BNGT04(RT73) DNA in a sampleand compositions thereof.

BACKGROUND OF THE INVENTION

Canola is an important oil crop in many areas of the world. The methodsof biotechnology have been applied to canola for improvement of theagronomic traits and the quality of the product. A method of introducingtransgenes into Brassica species is demonstrated in U.S. Pat. No.5,463,174. One such agronomic trait important in canola production isherbicide tolerance, in particular, tolerance to glyphosate herbicide.This trait has been introduced into canola plants and is a successfulproduct now used in canola production. The expression of foreign genesin plants is known to be influenced by their chromosomal position,perhaps due to chromatin structure (e.g., heterochromatin) or theproximity of transcriptional regulation elements (e.g., enhancers) closeto the integration site (Weising et al., Ann. Rev. Genet 22:421-477,1988). For this reason, it is often necessary to screen a large numberof events in order to identify an event characterized by optimalexpression of a introduced gene of interest. For example, it has beenobserved in plants and in other organisms that there may be a widevariation in levels of expression of an introduced genes among events.There may also be differences in spatial or temporal patterns ofexpression, for example, differences in the relative expression of atransgene in various plant tissues, that may not correspond to thepatterns expected from transcriptional regulatory elements present inthe introduced gene construct. For this reason, it is common to producehundreds to thousands of different events and screen those events for asingle event that has desired transgene expression levels and patternsfor commercial purposes. An event that has desired levels or patterns oftransgene expression is useful for introgressing the transgene intoother genetic backgrounds by sexual outcrossing using conventionalbreeding methods. Progeny of such crosses maintain the transgeneexpression characteristics of the original transformant. This strategyis used to ensure reliable gene expression in a number of varieties thatare well adapted to local growing conditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the premarket approval and labeling of foods derived fromrecombinant crop plants, for example. It is possible to detect thepresence of a transgene by any well known nucleic acid detection methodsuch as the polymerase chain reaction (PCR) or DNA hybridization usingnucleic acid probes. These detection methods generally focus onfrequently used genetic elements, such as promoters, terminators, markergenes, etc. As a result, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct unless the sequence of chromosomal DNAadjacent to the inserted DNA (“flanking DNA”) is known. Anevent-specific PCR assay is discussed, for example, by Windels et al.(Med. Fac. Landbouww, Univ. Gent 64/5b:459-462, 1999), who identifiedglyphosate tolerant soybean event 40-3-2 by PCR using a primer setspanning the junction between the insert and flanking DNA, specificallyone primer that included sequence from the insert and a second primerthat included sequence from flanking DNA.

This invention relates to the glyphosate herbicide tolerant canola(Brassica napus) plant PV-BNGT04(RT73) sold in the U.S.A. and othercountries under the name Roundup Ready® canola and to the DNA moleculescontained in these canola plants that are useful in detection methodsfor Roundup Ready® canola and progeny thereof.

SUMMARY OF THE INVENTION

According to one aspect of the invention, DNA sequences that comprise apolynucleotide of sufficient length of polynucleotides homologous to thetransgene portion of the DNA sequence of SEQ ID NO:7 or complementsthereof, and a similar length of polynucleotides homologous to theflanking canola DNA sequence of SEQ ID NO:7 or complements thereof,wherein the polynucleotide is useful as a DNA primer in DNAamplification methods or DNA hybridization methods.

According to another aspect of the invention, DNA sequences thatcomprise a sufficient length of polynucleotides of the transgene portionof the DNA sequence of SEQ ID NO:8 or complements thereof, and a similarlength of polynucleotides homologous to the flanking canola DNA sequenceof SEQ ID NO:8 or complements thereof, wherein the polynucleotide isuseful as a DNA primer in DNA amplification methods or DNA hybridizationmethods.

According to an aspect of the invention, compositions and methods areprovided for detecting the presence of the transgene/genomic insertionregion from a canola plant designated PV-BNGT04(RT73) and plants andseeds thereof. DNA sequences are provided that comprise at least onetransgene/genomic insertion region junction sequence of PV-BNGT04(RT73)identified as SEQ ID NO:5 and SEQ ID NO:6, and complements thereof;wherein an insertion region junction sequence is a DNA polynucleotidesequence that spans the heterologous DNA inserted into the canola genomeand the endogenous DNA of the canola genome at the insertion site and isdiagnostic for the event.

According to another aspect of the invention, a DNA sequence thatcomprises the novel transgene/genomic insertion region, SEQ ID NO:7 isan aspect of this invention. Included are DNA sequences that comprise asufficient length of polynucleotides of transgene insert sequence and asufficient length of polynucleotides of canola genomic sequence fromcanola plant PV-BNGT04(RT73) of SEQ ID NO:7 that are useful as DNAprimer polynucleotides for the production of an amplicon productdiagnostic for canola plant PV-BNGT04(RT73). The DNA primerpolynucleotides comprise a primer set. Therefore the invention alsoincludes the primer set and the amplicons produced by primers setswherein the DNA primer polynucleotides are homologous or complementaryto SEQ ID NO:7.

According to another aspect of the invention, a DNA sequence thatcomprises the novel transgene/genomic insertion region, SEQ ID NO:8 isan aspect of this invention. Included are DNA sequences that comprise asufficient length of polynucleotides of transgene insert sequence and asufficient length of polynucleotides of canola genomic sequence fromcanola plant PV-BNGT04(RT73) of SEQ ID NO:8 that are useful as DNAprimer polynucleotides for the production of an amplicon productdiagnostic for canola plant PV-BNGT04(RT73). The DNA primerpolynucleotides comprise a primer set. Therefore the invention alsoincludes the primer set and the amplicons produced by primers setswherein the DNA primer polynucleotides are homologous or complementaryto SEQ ID NO:8.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding to the canola event PV-BNGT04(RT73) eventin a sample are provided. Such methods comprise: (a) contacting a DNAsample with a primer set, that when used in a nucleic acid amplificationreaction with DNA from canola event PV-BNGT04(RT73) produces an ampliconthat is diagnostic for canola event PV-BNGT04(RT73); (b) performing anucleic acid amplification reaction, thereby producing the amplicon; and(c) detecting the amplicon.

According to another aspect of the invention, methods of detecting thepresence of a DNA corresponding to the PV-BNGT04(RT73) event in asample, such methods comprising: (a) contacting the sample comprisingDNA with a probe that hybridizes under stringent hybridizationconditions with DNA from canola event PV-BNGT04(RT73) and does nothybridize under the stringent hybridization conditions with a controlcanola plant (non-PV-BNGT04(RT73); and (b) subjecting the sample andprobe to stringent hybridization conditions; and (c) detectinghybridization of the probe to the DNA.

According to another aspect of the invention, methods of producing acanola plant that tolerates application of glyphosate are provided thatcomprise the steps of: (a) sexually crossing a first parental canolaline comprising the expression cassettes of the present invention, whichconfers tolerance to application of glyphosate, and a second parentalcanola line that lacks the glyphosate tolerance, thereby producing aplurality of progeny plants; and (b) selecting a progeny plant by theuse of molecular markers SEQ ID NO:5 and SEQ ID NO:6, or complementsthereof in a marker assisted breeding method. Such methods mayoptionally comprise the further step of back-crossing the progeny plantto the second parental canola line to producing a true-breeding canolaplant that tolerates application of glyphosate.

According to another aspect of the invention, methods of determining thezygosity of progeny of a cross with PV-BNGT04(RT73) are provided. Amethod that comprises contacting a sample consisting of canola DNA witha primer set comprising SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15,that when used in a nucleic-acid amplification reaction with genomic DNAfrom canola event PV-BNGT04(RT73), produces a first amplicon that isdiagnostic for canola event PV-BNGT04(RT73); and performing a nucleicacid amplification reaction, thereby producing the first amplicon; anddetecting the first amplicon; and contacting the sample comprisingcanola DNA with said primer set, that when used in a nucleic-acidamplification reaction with genomic DNA from canola plants produces asecond amplicon comprising the native canola genomic DNA homologous tothe canola genomic region of a transgene insertion identified as canolaevent PV-BNGT04(RT73); and performing a nucleic acid amplificationreaction, thereby producing the second amplicon; and detecting thesecond amplicon; and

comparing the first and second amplicons in a sample, wherein thepresence of both amplicons indicates the sample is heterozygous for thetransgene insertion.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,Genes V, Oxford University Press: New York, 1994. The nomenclature forDNA bases as set forth at 37 CFR §1.822 is used.

As used herein, the term “canola” means Brassica napus and includes allplant varieties that can be bred with canola, including wild Brassicaspecies.

As used herein, the term “comprising” means “including but not limitedto”.

“Glyphosate” refers to N-phosphonomethylglycine and its salts,Glyphosate is the active ingredient of Roundup® herbicide (MonsantoCo.). Treatments with “glyphosate herbicide” refer to treatments withthe Roundup®, Roundup Ultra® herbicide or any other herbicideformulation containing glyphosate. For the purposes of the presentinvention, the term “glyphosate” includes any herbicidally active formof N-phosphonomethylglycine (including any salt thereof) and other formsthat result in the production of the glyphosate anion in plants.Treatments with “glyphosate” refer to treatments with the Roundup® orRoundup Ultra® herbicide formulation, unless otherwise stated. Planttransformation and regeneration in tissue culture use glyphosate orsalts of glyphosate. Whole plant assays use formulated Roundup® orRoundup Ultra®. Additional formulations with herbicide activity thatcontain N-phosphonomethylglycine or any of its salts are herein includedas a glyphosate herbicide.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, i.e., a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety thatinclude the heterologous DNA. Even after repeated back-crossing to arecurrent parent, the inserted DNA and flanking DNA from the transformedparent is present in the progeny of the cross at the same chromosomallocation. The term “event” also refers to DNA from the originaltransformant comprising the inserted DNA and flanking genomic sequenceimmediately adjacent to the inserted DNA that would be expected to betransferred to a progeny that receives inserted DNA including thetransgene of interest as the result of a sexual cross of one parentalline that includes the inserted DNA (e.g., the original transformant andprogeny resulting from selfing) and a parental line that does notcontain the inserted DNA. A glyphosate tolerant canola plant can bebreed by first sexually crossing a first parental canola plantconsisting of a canola plant grown from the transgenic canola plantderived from transformation with the expression cassettes of the presentinvention that tolerates application of glyphosate herbicide, and asecond parental canola plant that lacks the tolerance to glyphosateherbicide, thereby producing a plurality of first progeny plants; andthen selecting a first progeny plant that is tolerant to application ofglyphosate herbicide; and selfing the first progeny plant, therebyproducing a plurality of second progeny plants; and then selecting fromthe second progeny plants a glyphosate herbicide tolerant plant. Thesesteps can further include the back-crossing of the first glyphosatetolerant progeny plant or the second glyphosate tolerant progeny plantto the second parental canola plant or a third parental canola plant,thereby producing a canola plant that tolerates the application ofglyphosate herbicide.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr, inBreeding Methods for Cultivar Development, Wilcox J. ed., AmericanSociety of Agronomy, Madison Wis. (1987).

Canola varieties containing genomic DNA from canola eventPV-BNGT04(RT73) has been introduced into commercial germplasm and iscommercially available in Roundup Ready® Canola varieties.

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of genomic DNA from canola eventPV-BNGT04(RT73) whether from a canola plant or from a sample thatincludes DNA from the event. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that bind specifically to a targetDNA sequence and can be used to detect the presence of that target DNAsequence.

“Primers” are isolated polynucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. Primerpairs of the present invention refer to their use for amplification of atarget nucleic acid sequence, e.g., by the polymerase chain reaction(PCR) or other conventional nucleic-acid amplification methods.

Probes and primers are generally 11 nucleotides or more in length,preferably 18 nucleotides or more, more preferably 24 nucleotides ormore, and most preferably 30 nucleotides or more. Such probes andprimers hybridize specifically to a target sequence under highstringency hybridization conditions. Preferably, probes and primersaccording to the present invention have complete sequence identity withthe target sequence, although probes differing from the target sequenceand that retain the ability to hybridize to target sequences under highstringency conditions may be designed by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, N.Y., 1992 (with periodic updates) (hereinafter,“Ausubel et al., 1992”); and Innis et al., PCR Protocols: A Guide toMethods and Applications, Academic Press: San Diego, 1990. PCR-primerpairs (a primer set) can be derived from a known sequence, for example,by using computer programs intended for that purpose such as Primer(Version 0.5, @1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

Primers and probes based on the flanking genomic DNA and insertsequences disclosed herein can be used to confirm (and, if necessary, tocorrect) the disclosed sequences by conventional methods, e.g., byre-cloning and sequencing such sequences.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA sequence. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic event in a sample.Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure andare of sufficient length to maintain this structure under highstringency conditions. A nucleic acid molecule is said to be the“complement” of another nucleic acid molecule if they exhibit completecomplementarity. As used herein, molecules are said to exhibit “completecomplementarity” when every nucleotide of one of the molecules iscomplementary to a nucleotide of the other. Two molecules are said to be“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are said to be “complementary” if they can hybridize to oneanother with sufficient stability to permit them to remain annealed toone another under conventional “high-stringency” conditions.Conventional stringency conditions are described by Sambrook et al.,1989, and by Haymes et al., In: Nucleic Acid Hybridization, A PracticalApproach, IRL Press, Washington, D.C. (1985), Departures from completecomplementarity are therefore permissible, as long as such departures donot completely preclude the capacity of the molecules to form adouble-stranded structure. In order for a nucleic acid molecule to serveas a primer or probe it need only be sufficiently complementary insequence to be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In a preferredembodiment, a nucleic acid of the present invention will specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NO:5 and SEQ ID NO:6 or complements thereof or fragments of eitherunder moderately stringent conditions, for example at about 2.0×SSC andabout 65° C. In a particularly preferred embodiment, a nucleic acid ofthe present invention will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NO:5 and SEQ ID NO:6 orcomplements or fragments of either under high stringency conditions. Inone aspect of the present invention, a preferred marker nucleic acidmolecule of the present invention has the nucleic acid sequence setforth SEQ ID NO: 5 and SEQ ID NO:6 or complements thereof or fragmentsof either. In another aspect of the present invention, a preferredmarker nucleic acid molecule of the present invention shares between 80%and 100% or 90% and 100% sequence identity with the nucleic acidsequence set forth in SEQ ID NO:5 and SEQ ID NO:6 or complement thereofor fragments of either. In a further aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 95% and 100% sequence identity with the sequence set forth inSEQ ID NO:5 and SEQ ID NO:6 or complement thereof or fragments ofeither. SEQ ID NO:5 and SEQ ID NO:6 may be used as markers in plantbreeding methods to identify the progeny of genetic crosses similar tothe methods described for simple sequence repeat DNA marker analysis, in“DNA markers: Protocols, applications, and overviews: (1997) 173-185,Cregan, et al., eds., Wiley-Liss N.Y.; all of which is hereinincorporated by reference in its' entirely. The hybridization of theprobe to the target DNA molecule can be detected by any number ofmethods known to those skilled in the art, these can include, but arenot limited to, fluorescent tags, radioactive tags, antibody based tags,and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thecanola plant resulting from a sexual cross contains transgenic eventgenomic DNA from the canola plant of the present invention, DNAextracted from a canola plant tissue sample may be subjected to anucleic acid amplification method using a primer pair that includes aprimer derived from flanking sequence in the genome of the plantadjacent to the insertion site of inserted heterologous DNA, and asecond primer derived from the inserted heterologous DNA to produce anamplicon that is diagnostic for the presence of the event DNA. Theamplicon is of a length and has a sequence that is also diagnostic forthe event. The amplicon may range in length from the combined length ofthe primer pairs plus one nucleotide base pair, or plus about fiftynucleotide base pairs, or plus about two hundred-fifty nucleotide basepairs, or plus about three hundred-fifty nucleotide base pairs or more.Alternatively, a primer pair can be derived from flanking genomicsequence on both sides of the inserted DNA so as to produce an ampliconthat includes the entire insert nucleotide sequence. A member of aprimer pair derived from the plant genomic sequence may be located adistance from the inserted DNA sequence, this distance can range fromone nucleotide base pair up to about twenty thousand nucleotide basepairs. The use of the term “amplicon” specifically excludes primerdimers that may be formed in the DNA thermal amplification reaction.

Nucleic acid amplification can be accomplished by any of the variousnucleic acid amplification reaction methods known in the art, includingthe polymerase chain reaction (PCR). A variety of amplification methodsare known in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic Press, San Diego, 1990. PCRamplification methods have been developed to amplify up to 22 kb ofgenomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc.Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as othermethods known in the art of DNA amplification may be used in thepractice of the present invention. The sequence of the heterologous DNAinsert or flanking sequence from canola event PV-BNGT04(RT73) can beverified (and corrected if necessary) by amplifying such sequences fromthe event using primers derived from the sequences provided hereinfollowed by standard DNA sequencing methods applied to the PCR ampliconor to isolated cloned transgene/genomic DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:4167-4175, 1994) where an DNA oligonucleotide isdesigned which overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microwell plate. Following PCR of the region of interest(using one primer in the inserted sequence and one in the adjacentflanking genomic sequence), a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking genomic sequence) and incubated in the presenceof a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. DNTPs are added individually and theincorporation results in a light signal which is measured. A lightsignal indicates the presence of the transgene insert/flanking sequencedue to successful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedwhich overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps thegenomic flanking and insert DNA junction. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996) Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization.

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art of DNA detection. The kitsare useful for the identification of canola event PV-BNGT04(RT73) DNA ina sample and can be applied to methods for breeding canola plantscontaining PV-BNGT04(RT73) DNA. The kits contain DNA sequenceshomologous or complementary to SEQ ID NO:7 or SEQ ID NO:8 or to DNAsequences homologous or complementary to DNA contained in the transgenegenetic elements of PV-BNGT04(RT73) DNA, these DNA sequences can be usedin DNA amplification reactions or as probes in a DNA hybridizationmethod. The sequences of the transgene genetic elements contained inPV-BNGT04(RT73) DNA consists of the Figwort mosaic promoter (U.S. Pat.No. 5,378,619, herein incorporated by reference in its entirety)operably connected to an Arabidopsis EPSPS chloroplast transit peptide(At.EPSPS:CTP2, U.S. Pat. No. 5,633,435, herein incorporated byreference in its entirety), operably connected to a glyphosateoxidoreductase gene (U.S. Pat. No. 5,776,760, herein incorporated byreference in its entirety), operably connected to the 3′ terminationregion from pea ribulose 1,5-bisphosphate carboxylase E9 (Coruzzi, etal., EMBO J. 3:1671-1679, 1984, herein incorporated by reference in itsentirety), and in tandem orientation, the Figwort mosaic promoter (U.S.Pat. No. 5,378,619) operably connected to an Arabidopsis EPSPSchloroplast transit peptide (At.EPSPS:CTP2), operably connected to aglyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS)from Agrobacterium sp. strain CP4 (AGRTU.aroA:CP4 EPSPS, U.S. Pat. No.5,633,435, herein incorporated by reference in its entirety), operablyconnected to the 3′ termination region from pea ribulose1,5-bisphosphate carboxylase E9. DNA molecules useful as primers in DNAamplification methods can be derived from the sequences of the geneticelements of the transgene insert contained in PV-BNGT04(RT73) canolaevent. These primer molecules can be used as part of a primer set thatalso includes a DNA primer molecule derived from the genome ofPV-BNGT04(RT73) event flanking the transgene insert.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1

DNA from PV-BNGT04(RT73) transgenic canola event (hence forth referredto as RT73 event) was extracted from canola seeds containing the RT73event and nontransgenic canola line Golden Boy. The DNA was isolatedfrom seed tissue using Qiagen's DNeasy Plant Miniprep Kit according tothe manufacturer's instructions (Qiagen Corp. Valencia Calif.).

PCR of the genomic sequences flanking the 5′ end of the insert in RT73event was performed using primer 1 sequence (SEQ ID NO: 1, 5′CTTGTTGAGGCTTTGGACTGAGAAT 3′) derived from the 5′ genomic flankingsequence paired with primer 2 sequence (SEQ ID NO:2, 5′CGCTCTCTCTTAGTTTTGAAATACA 3′) or the complements thereof, located in theinsert transgene sequence adjacent to the right border region of theT-DNA. The PCR analysis for the genomic sequence flanking the 3′ end ofthe RT73 event insert was conducted using primer 3 sequence (SEQ IDNO:3, 5′ TGAATGTAGACACGTCGAAATAAAGATT 3′) located in the transgenesequence coupled with primer 4 (SEQ ID NO:4, 5′TACTTGAAGCACACGACACTGTAATTC 3′) or the complements thereof, derived fromthe 3′ genomic flanking sequence. The PCR were performed using ˜50 ng ofRT73 or nontransgenic genomic DNA template in a 50 μl reaction volume.Each reaction contained 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 200 μMof each DNTP, 0.4 mM each primer, and 2.5 units of RedTaq DNApolymerase. The PCR were performed under the following cyclingconditions: 1 cycle at 94° C. for 3 minutes; 35 cycles (or 30 cycles forthe 3′-flank analysis) at 94° C. for 30 s, 57.5° C. (or 55° C. for the3′-flank analysis) for 30 s and 72° C. for 1.5 minutes; 1 cycle at 72°C. for 10 minutes. Twenty microliters of each reaction were separated ona 1.5% agarose gel. The PCR products were visualized by ethidium bromidestaining under UV illumination.

PCR products of the expected sizes representing the 5′ and3′transgene/genomic sequences were isolated by separation of the PCRproducts on a 2.0% agarose gel by electrophoresis. PCR products,representing 5′ regions that span the junction between the RT73 eventtransgenic insertion and the neighboring flanking canola genome DNAsequence were purified by agarose gel electrophoresis followed byisolation from the agarose matrix using the QIAquick Gel Extraction Kit(catalog #28704, Qiagen Inc., Valencia, Calif.). The purified PCRproducts were then sequenced with by DNA sequence analysis (ABI Prism™377, PE Biosystems, Foster City, Calif. and DNASTAR sequence analysissoftware, DNASTAR Inc., Madison, Wis.).

The DNA sequence was determined for a 353 nucleotide base pair segmentrepresenting the 5′ transgene/genomic insert sequence of canola RT73event and identified in SEQ ID NO:7. The DNA sequence was determined fora 474 nucleotide base pair segment representing the 3′ transgene/genomicinsert sequence of canola RT73 event and identified in SEQ ID NO:8.

The junction sequences, SEQ ID NO:5 (5′ATCAGTGTTCGACTTTTT 3′) and SEQ IDNO:6 (5′ GACATGAAGATCATCCTC 3′) are novel DNA sequences in RT73 eventand are diagnostic for canola plant RT73 event and progeny thereof. Thejunction sequences in SEQ ID NO:5 and SEQ ID NO:6 represent 9polynucleotides on each side of an insertion site of the transgenesequence fragment and canola genomic DNA, longer or shorterpolynucleotide sequences can be selected from SEQ ID NO:7 or SEQ ID NO:8that represent the junction sequences. SEQ ID NO:5 is found atnucleotide positions 199-216 of SEQ ID NO:7, and the junction sequenceSEQ ID NO:6 is located at nucleotide positions 228-245 of SEQ ID NO:8,representing the transgene/genomic insert junction sequences in RT73event and progeny thereof.

Example 2

DNA event primer pairs are used to produce an amplicon diagnostic forRT73 event. These event primer pairs include, but are not limited to SEQID NO:9 (5′ CATGTAGATTTCCCGGACATGAAG 3′) and SEQ ID NO:10(5′GTGTGAATTACAGTGTCGTGTGC 3′) or the complements thereof. The ampliconproduced by SEQ ID NO:9 and SEQ ID NO: 10 is about 265 polynucleotides.In addition to these primer pairs, any primer pair derived from SEQ IDNO:7 or SEQ ID NO:8 or the complements thereof, that when used in a DNAamplification reaction produces an amplicon diagnostic for RT73 event isan aspect of the present invention. The amplification conditions forthis analysis is illustrated in Table 1 and Table 2, however, anymodification of these methods that use DNA primers to produce anamplicon diagnostic for RT73 event is within the ordinary skill of theart. In addition, a control primer pair (SEQ ID NO: 11, 5′GTTACAGATGAAGTTCGGGACG 3′ and SEQ ID NO:12, 5′ GCAAGAACTGGCTCTCATTGTG3′) for amplification of an endogenous canola gene (FatA) is included asan internal standard for the reaction conditions and produces anamplicon of approximately 595 polynucleotides. The analysis of RT73event plant tissue sample should include a positive tissue control fromRT73 event, a negative control from a canola plant that is not RT73event, and a negative control that contains no template canola DNA.Additional primer sequences can be selected from SEQ ID NO:7 and SEQ IDNO:8 by those skilled in the art of DNA amplification methods, andconditions optimized for the production of an amplicon that may differfrom the methods shown in Table 1 and Table 2, but result in an amplicondiagnostic for RT73. The use of these DNA primer sequences withmodifications to the methods of Table 1 and 2 are within the scope ofthe invention. The amplicon produced by the use of at least one primersequence derived from SEQ ID NO:7, or at least one primer sequencederived from SEQ ID NO:8 that when used in a PCR method produces anamplicon diagnostic for RT73 event can be used in the described methodsand is an aspect of the invention. The production of the RT73 eventamplicon can be performed by using a Stratagene Robocycler, MJ Engine,Perkin-Elmer 9700, or Eppendorf Mastercycler Gradient thermocycler asshown in Table 2, or by methods and apparatus known to those skilled inthe art.

TABLE 1 PCR procedure and reaction mixture for the confirmation of RT735′ transgene insert/genomic junction region. Step Reagent AmountComments 1 Nuclease-free water add to final volume — of 20 μl 2 10Xreaction buffer 2.0 μl 1X final (with MgCl₂) concentration of buffer,1.5 mM final concentration of MgCl₂ 3 10 mM solution of dATP, 0.4 μl 200μM final dCTP, dGTP, and dTTP concentration of each dNTP 4 Event primer9 (SEQ ID NO: 9 0.2 μl 0.1 μM final resuspended in 1X TE buffer orconcentration nuclease-free water to a concentration of 10 μM) 5 Eventprimer 10 (SEQ ID 0.2 μl 0.1 μM final NO: 10 resuspended in 1X TEconcentration buffer or nuclease-free water to a concentration of 10 μM)6 Control primer 11 (SEQ ID 0.2 μl 0.1 μM final NO: 11 resuspended in 1XTE concentration buffer or nuclease-free water to a concentration of 10(μM) 7 Control primer 12 (SEQ ID 0.2 μl 0.1 μM final NO: 12 resuspendedin 1X TE concentration buffer or nuclease-free water to a concentrationof 10 μM) 8 RNase, DNase free (500 μg/ml) 0.1 μl 50 ng/reaction 9 REDTaqDNA polymerase 1.0 μl (recommended to 1 unit/reaction (1 unit/μl) switchpipets prior to next step) 10 Extracted DNA (template): — Samples to beanalyzed: individual leaves 10-200 ng of genomic DNA pooled leaves(maximum 200 ng of genomic DNA of 10 leaves/pool) Negative control 50 ngof non-transgenic canola genomic DNA Negative control no template DNA(solution in which DNA was resuspended) Positive control 50 ng of RT73genomic DNA

Gently mix and, if needed (no hot top on thermocycler), add 1-2 drops ofmineral oil on top of each reaction. Proceed with the PCR in aStratagene Robocycler, MJ Engine, Perkin-Elmer 9700, or EppendorfMastercycler Gradient thermocycler using the following cyclingparameters (Table 2). The MJ Engine or Eppendorf Mastercycler Gradientthermocycler should be run in the calculated mode. Run the Perkin-Elmer9700 thermocycler with the ramp speed set at maximum.

TABLE 2 Thermocycler conditions Cycle No. Settings: StratageneRobocycler 1 94° C. 3 minutes 34 94° C. 1 minute 64° C. 1 minute 72° C.1 minute and 30 seconds 1 72° C. 10 minutes Cycle No. Settings: MJEngine or Perkin-Elmer 9700 1 94° C. 3 minutes 34 94° C. 30 seconds 64°C. 30 seconds 72° C. 1 minute 1 72° C. 10 minutes Cycle No. Settings:Eppendorf Mastercycler Gradient 1 94° C. 3 minutes 34 94° C. 15 seconds64° C. 15 seconds 72° C. 1 minute 1 72° C. 10 minutes

Example 3

the methods used to identify heterozygous from homozygous canola progenycontaining RT73 event DNA are described in the zygosity assay in Table 3and Table 4. The DNA primers used in the zygosity assay are: SEQ ID NO:13, 5′ CATGTAGATTTCCCGGACATGAAG 3′; SEQ ID NO: 14, 5′GTGTGAATTACAGTGTCGTGTGC 3′; SEQ ID NO: 15, 5′GAGATGTATTTCAAAACTAAGAGAGAGC 3′.

SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 when used in these reactionmethods produce a DNA amplicon of 409 polynucleotide base pairs (bps)for non-transgenic canola, two DNA amplicons of 409 bps and 265 bps forheterozygous canola containing RT73 event DNA, and a DNA amplicon of 265bp for homozygous canola containing RT73 event DNA. The controls forthis analysis should include a positive control from homozygous andheterozygous canola containing RT73 event DNA, a negative control fromnon-transgenic canola, and a negative control that contains no templateDNA. This assay is optimized for use with a Stratagene Robocycler, MJEngine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradientthermocycler. Other methods and apparatus known to those skilled in theart that produce amplicons that identify the zygosity of the progeny ofcrosses made with RT73 event canola plants is within the skill of theart.

TABLE 3 Zygosity assay reaction solutions Step Reagent Amount Comments 1Nuclease-free water add to 20 μl — final volume 2 10X reaction buffer  2 μl 1.5 mM final (with MgCl₂) concentration of MgCl₂ 3 10 mM solutionof dATP, 0.4 μl 200 μM final dCTP, dGTP, and dTTP concentration of eachdNTP 4 SEQ ID NO: 13 primer 0.5 μl 0.25 μM final resuspended in 1X TEconcentration buffer or nuclease-free water to a concentration of 10 μM)5 SEQ ID NO: 14 primer 0.8 μl 0.4 μM final resuspended in 1X TEconcentration buffer or nuclease-free water to a concentration of 10 μM)6 SEQ ID NO: 15 primer 0.3 μl 0.15 μM final resuspended in 1X TEconcentration buffer or nuclease-free water to a concentration of 10 μM)7 REDTaq DNA polymerase 1.0 μl (recommended to 1 unit/reaction (1unit/μl) switch pipets prior to next step) 8 Extracted DNA (template): —Samples to be analyzed 10-200 ng of genomic (individual leaves) DNANegative control 10-200 ng of non- transgenic canola genomic DNANegative control no DNA template (solution in which DNA was resuspended)Heterozygous 10-200 ng of genomic Positive control DNA from known eventRT73 heterozygous canola Homozygous 10-200 ng of genomic Positivecontrol DNA from known event RT73 homozygous canola

Gently mix and, if needed (no hot top on thermocycler), add 1-2 drops ofmineral oil on top of each reaction. Proceed with the PCR in aStratagene Robocycler, MJ Engine, Perkin-Elmer 9700, or EppendorfMastercycler Gradient thermocycler using the following cyclingparameters (Table 4). The MJ Engine or Eppendorf Mastercycler Gradientthermocycler should be run in the calculated mode. Run the Perkin-Elmer9700 thermocycler with the ramp speed set at maximum.

TABLE 4 Zygosity assay thermocycler conditions Cycle No. Settings:Stratagene Robocycler 1 94° C. 3 minutes 38 94° C. 1 minute 54° C. 1minute 72° C. 1 minute and 30 seconds 1 72° C. 10 minutes Cycle No.Settings: MJ Engine or Perkin-Elmer 9700 1 94° C. 3 minutes 38 94° C. 30seconds 54° C. 30 seconds 72° C. 1 minute and 30 seconds 1 72° C. 10minutes Cycle No. Settings: Eppendorf Mastercycler Gradient 1 94° C. 3minutes 38 94° C. 15 seconds 54° C. 15 seconds 72° C. 1 minute and 30seconds 1 72° C. 10 minutes

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. A DNA molecule comprising SEQ ID NO:5.
 2. A primer pair of DNA molecules, wherein a first primer of said primer pair comprises at least 11 contiguous nucleotides from nucleotide 208-353 of the transgene region of SEQ ID NO:7 or complements thereof and a second primer of said primer pair comprises at least 11 contiguous nucleotides from nucleotides 1-207 of the canola genomic DNA sequence of SEQ ID NO:7 or complements thereof and the pair of DNA molecules when used together in a DNA amplification method produces a diagnostic amplicon comprising SEQ ID NO:5 or complements thereof for canola event PV-BNGT04(RT73).
 3. A method of detecting the presence of genomic/transgene DNA of canola PV-BNGT04(RT73) event comprising SEQ ID NO:5, in a sample, the method comprising: (a) contacting the sample comprising canola DNA with a primer pair of claim 2; and (b) performing a nucleic acid amplification reaction, thereby producing an amplicon comprising SEQ ID NO:5; and (c) detecting the amplicon.
 4. The DNA molecule of claim 1, wherein the DNA molecule is SEQ ID NO:7.
 5. A DNA detection kit comprising at least one DNA molecule of sufficient length of contiguous nucleotides homologous or complementary to SEQ ID NO:7 to function as a DNA probe specific for canola event PV-BNGT04(RT73), wherein said DNA molecule comprises SEQ ID NO:5.
 6. A method of detecting the presence of a genomic/transgene DNA molecule of PV-BNGT04(RT73) comprising SEQ ID NO:5, in a sample, the method comprising: (a) contacting the sample comprising canola DNA with a polynucleotide probe that hybridizes under stringent hybridization conditions with DNA from canola event PV-BNGT04(RT73) and does not hybridize under the stringent hybridization conditions with a non-PV-BNGT04(RT73) canola plant DNA, wherein the probe comprises the DNA molecule of claim 1; and (b) subjecting the sample and probe to stringent hybridization conditions; (c) detecting hybridization of the probe to the DNA.
 7. The DNA molecule of claim 1 wherein the DNA molecule is SEQ ID NO:5.
 8. A method of selecting a canola plant or seeds thereof comprising a glyphosate tolerant trait, the method comprising the steps of: (a) obtaining at least one canola plant; (b) detecting SEQ ID NO:5 within said canola plant; and (c) selecting a canola plant comprising SEQ ID NO:5.
 9. The method of claim 8 further comprising the step of: (d) producing seed from said canola plant of step (c).
 10. A DNA detection kit comprising the primer pair of claim
 2. 